Molecular Biochemical Study on the Cyanobacterial ndh (NADH dehydrogenase) Gene Products

蓝藻ndh（NADH脱氢酶）基因产物的分子生化研究

• 批准号: 06640840
• 负责人: TAKAHASHI Yasuhiro
• 金额: $ 1.41万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06640840/
• 关键词: Cyanobacteria; ndh genes; NAD (P) H dehydrogenase; NAD (P) H脱水素酵素; NAD(P)H 脱水素酵素

Eleven ndh genes (ndhA-K) have been identified in chloroplast and cyanobacterial genomes on the basis of sequence similarity to the subunits of mitochondrial and bacterial NADH : ubiquinone oxidoreductase. These findings have been taken to indicate the presence of an enzyme related to the mitochondrial and bacterial enzymes, possibly an NAD (P) H : plastoquinone oxidoreductase ; however, the active enzyme complex has not yet been isolated. In order to clarify its physiological significance, several attempts have been made using the filamentous cyanobacterium Plectonema boryanum, a facultative chemoheterotroph.Three essential ndh genes are missing from the plastid genomes. They are 51 (FP), 24 (FP), and 75 (IP) subunits of bovine complex I,which constitute a subcomplex involved in the first part of electron pathway from NADH to intermediate acceptors. Cyanobacterial DNA fragment was cloned after PCR amplification, which shows sequence similarity to the mitochondrial 51 (FP) subunit. Attempts to inactivate the ndhA,ndhI,ndhG,or ndhE gene of P.boryanum have been unsuccessful under both normal and high-CO2 growth conditions, suggesting an essential role in this organism. In order to identify ndh related genes, we have developed a system for mutagenesis using a non-replicating plasmid pTAF10 which was constructed from pUC12, the neo gene, and OMEGA fragment. Genomic fragments partially digested by Sau3A were ligated into pTAF10, introduced into the cells by electroporation and kanamycin resistant mutants were obtained after single cross-over recombination. Insertion of the entire plasmid into the chromosome may interrupt the gene or disturb the expression of flanking genes due to the terminator sequence in the OMEGA fragment. Screening for the defect in chemoheterotrophic growth resulted in the isolation of dozens of mutants which are now analyzed.

根据与线粒体和细菌NADH：泛醌氧化还原酶亚基的序列相似性，已在叶绿体和蓝藻基因组中鉴定出11个NDH基因(NDHA-K)。这些发现表明存在一种与线粒体和细菌酶有关的酶，可能是一种NAD(P)H：质醌氧化还原酶；然而，活性酶复合体尚未被分离出来。为了阐明其生理意义，人们利用兼性化学异养的丝状蓝藻Plectonema boryanum进行了几次尝试。它们是牛复合体I的51(FP)、24(FP)和75(IP)亚基，它们构成了参与NADH到中间受体的电子通路第一部分的亚复合体。经PCR扩增后克隆到蓝藻DNA片段，与线粒体51(FP)亚基序列具有相似性。在正常和高CO2生长条件下，灭活P.boryanum的NDHA、ndhI、ndhG或ndhE基因的尝试都没有成功，这表明它在这个生物体中发挥着重要的作用。为了鉴定NDH相关基因，我们建立了一个利用pUC12、neo基因和omega片段构建的非复制型质粒pTAF10的突变体系。将Sau3A部分消化的基因组片段连接到pTAF10中，通过电穿孔的方法导入细胞，通过单交叉重组获得卡那霉素抗性突变体。由于omega片段中的终止子序列，整个质粒插入到染色体中可能会中断基因或干扰侧翼基因的表达。对化学异养生长缺陷的筛选导致分离出数十个突变体，现在正在分析这些突变体。

项目成果

Hiroshi Matsubara: "Three Iron-Sulfur Proteins Encoded by Three ORFs in Chloroplasts and Cyanobacteria." Photosyn. Res.46. 107-115 (1995)

Hiroshi Matsubara：“叶绿体和蓝藻中三个 ORF 编码的三种铁硫蛋白。”

南善子: "広く生体反応に関わる鉄硫黄蛋白質" 生物物理. 35. 7-12 (1995)

Yoshiko Minami：“广泛参与生物反应的铁硫蛋白”生物物理学 35. 7-12 (1995)。

Hiroshi Matsubara, Hirozo Oh-oka, Yasuhiro Takahashi, Yuichi Fujita: "Three Iron-Sulfur Proteins Encoded by Three ORFs in Chloroplasts and Cyanobacteria." Photosyn.Res.46 (1-2). 107-115 (1995)

Hiroshi Matsubara、Hirozo Oh-​​oka、Yasuhiro Takahashi、Yuichi Fujita：“叶绿体和蓝藻中三个 ORF 编码的三种铁硫蛋白。”

Yasuhiro Takahashi: "Cyanobacterial Mutagenesis Mediated by Ω-Containing Plasmid." (発表予定).

Yasuhiro Takahashi：“含 Ω 的质粒介导的蓝藻诱变。”（待公布）。

Yoshiko Minami, Yasuhiro Takahashi, Hiroshi Matsubara: "Iron-Sulfur Proteins Involved in Various Biological Reactions." Biophysics. 35 (2). 7-12 (1995)

Yoshiko Minami、Yasuhiro Takahashi、Hiroshi Matsubara：“参与各种生物反应的铁硫蛋白。”